This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Specific Aims: To clone ChChd3, ChChd6 and Mia40 into mammalian expression vectors with either GFP tags or Flag tags for visualization in an epifluorescent microscope. To introduce recombinant ChChd3, ChChd6, and Mia40 DNA into mammalian tissue culture cells and determine localization of the wild type proteins. To use bioinformatics techniques to determine potentially important mutation sites on the proteins. To introduce mutations in ChChd3, ChChd6, and Mia40 and monitor the effect on subcellular localization of the expressed proteins. To determine which amino acids in the proteins are necessary for targeting the proteins to the mitochondria. To explore other conserved sequence motifs through mutation and the effect on the proteins. Hypothesis: Proteins containing similar coiled coil-helix domains (CHCH) have a predicted mitochondrial localization. Mutation of key amino acids in the CHCH domain will disrupt targeting of the novel CHCH domain containing proteins ChChd3 and ChChd6.